NextSeq 500 sequencing; Determination of Transcription Start Sites (TSS) in Azotobacter vinelandii grown with iron, vanadium or molybdenum for 6 hours after nitrogen step-down
Determination of Transcription Start Sites (TSS) in Azotobacter vinelandii grown with iron, vanadium or molybdenum for 6 hours after nitrogen step-down
The cultures were mixed with 1/5 of stop solution (5% Phenol saturated with 0.1 M citrate pH 4.3, 95% ethanol) and then rapidly chilled on ice for 20 minutes. A. vinelandii was grown at 30°C and 250 rpm in NIL medium (containing 0.2 g/L MgCl2, 90 mg/L CaCl2, 0.8 g/L KH2PO4, 0.2 g/L K2HPO4, 14 mg/L Na2SO4, 120 mg/L Fe2(SO4)3 and 2.4 mg/L Na2MoO4) supplemented with 2% sucrose. To examine regulation of alternative nitrogenases in the absence of molybdenum, trace Mo (up to 8 ppb) was selectively removed by filtering NIL media through activated charcoal. A. vinelandii strains were grown under conditions of nitrogen access (NIL medium with ammonium acetate  25 mM) and subsequently subjected to nitrogen step down by centrifugation and resuspension to an O.D600 of 0.5 in NIL medium, supplemented with either Mo (1μM ), V (1μM) or no added metal (Fe-only conditions) and subsequently incubated for 6 hours prior to RNA extraction. Reads originating from processed RNAs (postfix PSS) were separated from those originating from primary transcripts (postfix TSS) on basis of different ligated tags by the sequencing contractor. RNA was purified using the TRI Reagent (Sigma #T9424) following manufacturer instructions. Genomic DNA was removed by three treatments with the TURBO DNA-free DNAse (Ambion #AM1907) according to the manufacturer instructions. cDNA synthesis was performed with SuperScript II Reverse Transcriptase (Invitrogen #18064014) using 0.1–1 μg of total RNA as recommended by the manufacturer. The concentration and integrity of the RNAs were verified with a Bioanalyer (Agilent) with the RNA 6000 nano kit (Agilent #5067-1511) following the manufacturer's instructions. Library preparation and sequencing were performed by the external contractor Vertis Biotechnologie AG as follows. 5' triphosphorylated RNA was capped with 3'- desthiobiotin-TEG-guanosine 5' triphosphate (DTBGTP) (NEB) using the vaccinia capping enzyme (VCE) (NEB) for reversible binding of biotinylated RNA species to streptavidin. Then biotinylated RNA species were captured on streptavidin beads and eluted to obtain the 5' fragment of the primary transcripts. The Cappable-seq enriched RNAs were first poly(A)-tailed using poly(A) polymerase. Then, the 5' Illumina TruSeq sequencing adapters, which carry sequence tags ATTACTCG and TCCGGAGA (in a proportion of 50% of each adapter), were ligated to the 5' mono-phosphate groups (5'P) of processsed transcripts. The sample was then treated with CapClip Acid Pyrophosphatase (Cellscript) in order to convert 5' triphosphate (5'PPP) structures into 5' monophosphate ends. To the newly formed 5'P groups were ligated the 5' Illumina TruSeq sequencing adapters, which carry sequence tag CGCTCATT and GAGATTCC (50% each). First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and the M-MLV reverse transcriptase. The resulting cDNA was PCR- amplified to about 10-20 ng/µl using a high fidelity DNA polymerase.